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Experimental Approaches For The Investigation Of Innate Immunity: The Human Innate Immunity Handbook

Experimental Approaches For The Investigation Of Innate Immunity: The Human Innate Immunity Handbook

Bucala Richard | Montgomery Ruth R

(2016)

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Book Details

Abstract

The recent explosion of information in innate immune pathways for recognition, effect or responses, and genetic regulation has given impetus to investigations into analogous pathways in the human immune response, which in turn has produced attendant insights into both normal physiology and immunopathology. This volume presents a compendium of methods and protocols for the investigation of human innate immunity with application to the study of normal immune function, immunosenescence, autoimmunity and infectious diseases. Among the topics covered are quantitative flow cytometry for Toll-like receptor expression and function; multidimensional single cell mass cytometry (CyTOF) in complex immune interactions and tumor immunity; imaging techniques such as Imagestream high resolution microscopy coupled to flow cytometry, immune cell infiltration of organotypic, biomimetic organs; high-throughput single cell secretion profiling; multiplexed transcriptomic profiling; microsatellite and microRNA methodologies, RNA interference; and the latest bioinformatics and biostatistical methodologies, including in-depth statistical modeling, genetic mapping, and systems approaches.

Table of Contents

Section Title Page Action Price
Contents v
Preface vii
List of Contributors ix
1. Assessment of Toll-Like Receptor Expression and Function by Flow Cytometry 1
1. Sample Preparation 2
1.1 Sample collection 2
1.2 Gradient centrifugation for isolation of PBMCs 2
2. Assessment of Human TLR Function 3
2.1 TLR Expression on monocytes and DCs 3
2.1.1 Surface Staining for TLR Expression 4
2.1.2 Intracellular staining for TLR expression 7
3. Assessment of Toll-like Receptor Function 7
3.1 TLR ligand induced co-stimulatory molecule expression in monocytes and dendritic cells 8
3.1.1 In-vitro stimulation of monocytes or DCs for co-stimulatory molecule expression 8
3.2. TLR ligand induced cytokine expression in monocytes and dendritic cells 8
3.2.1 In vitro stimulation of monocytes for cytokine production 8
3.2.2 In-vitro stimulation of dendritic cells for cytokine production 10
4. Comments on Analysis 11
4.1 Rational Approach 11
References 12
2. Dissecting Complex Cellular Systems with High Dimensional Single Cell Mass Cytometry 15
1. Introduction 15
2. The Mononuclear Phagocyte System 16
2.1. Early observations 16
2.2. Phenotype and function versus lineage identity 17
2.3. Complexity in biology and nomenclature: polarization subsets, M1 versus M2, TAMs, and MDSCs 17
3. Revisiting the Mononuclear Phagocyte System with High Dimensional Single Cell Analysis 20
3.1. Mass cytometry and machine learning 20
3.2. Mass cytometry’s contributions to myeloid biology 21
4. Future Directions 23
References 23
3. CyTOF: Single Cell Mass Cytometry for Evaluation of Complex Innate Cellular Phenotypes 27
1. Introduction 28
2. Materials 29
2.1 Antibody conjugation supplies 29
2.2 Mass cytometry labeling supplies 31
2.3 CyTOF mass cytometry running supplies 31
3. Methods 32
3.1 Antibody conjugation using maxpar metal labeling kits 32
3.2. Surface labeling of cells for mass cytometry 33
3.3 Running samples on a CyTOF mass cytometer 36
4. Notes 37
Acknowledgements 38
References 38
4. High-Throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity 41
1. Introduction 42
2. Materials 43
2.1 Reagents 43
2.2 Microfluidic supplies 44
2.3 Equipment 44
3. Procedure 44
3.1 Preparing the antibody barcode slide 44
3.1.1 Fabrication of new PDMS chip used for flowpatterning (Fig. 1). 44
3.1.2 Clean PDMS flow-patterning chip (if reusing) 45
3.1.3 Bond flow-patterning chip to glass slide 46
3.1.4 Flow patterning the antibody barcode array 46
3.2 Preparing the microchamber array 47
3.2.1 Fabrication of PDMS microchamber array (Fig. 1). 47
3.2.2 Condition the microchamber chip for cell culture 48
3.3 Cell Loading and stimulation (use sterile conditions) 48
3.3.1 Seeding adherent cells onto chip (Note 7) 48
3.3.2 Stimulating cells 49
3.3.3 Imaging microchamber array (note 8) 49
3.4 Immunoassay 50
3.4.1 Device disassembly 50
3.4.2 Developing the antibody barcode array 50
3.5 Data processing and analysis 51
3.5.1 Count cells 51
3.5.2 Analyze barcode intensities 51
3.5.3 Process raw data matrices 51
4. Notes 52
Acknowledgement 53
References 53
5. Analysis of Tissue Microenvironments Using Decellularized Mammalian Tissues 55
1. Introduction 55
2. Representative Results 56
2.1 Structure and composition of decellularized mammalian lungs 56
2.2 Decellularized lung scaffolds can be used to study macrophage:fibroblast crosstalk 60
3. Conclusion 60
4. Detailed Technical Information 62
4.1 Preparation of rat lung scaffold slices 62
4.2 Preparation of mouse lung scaffold slices 62
4.3 Mouse fibroblast coculture with mouse macrophages 63
References 63
6. Defining Innate Immune Pathways with Targeted RNAi Silencing 65
1. Introduction 66
2. Materials 67
2.1 Reagents 67
2.2 Equipment 67
3. Procedure 67
3.1 Transient transfection of siRNA 67
3.1.1 Design of siRNA sequence 67
3.1.2 Transfection of siRNA by lipofectamine 2000 68
3.2 Nucleofection of siRNA 68
3.2.1 Preparation of Peripheral Blood Mononuclear Cells (PBMCs) 69
3.2.2 Differentiation of macrophages 69
3.2.3 Nucleofection 70
3.3 Stable RNAi with shRNA lentivectors 70
3.3.1 Generating shRNA constructs 71
3.3.2 Production of lentiviral particles 72
3.3.3 Target cell infection and selection 72
4. Notes 73
Acknowledgement 73
References 73
7. ImageStream Methodologies for Flow Cytometry with High Resolution Microscopy 77
1. Introduction 77
2. Equipment Options and Selection 79
3. Software 80
4. Getting Started 81
4.1 Selecting dyes and staining 81
4.2 Compensation 83
4.3 Imaging rare cell populations 83
5. Applications 84
5.1 Transcription factor nuclear localization 84
5.2 Receptor co-localization and intracellular localization 84
5.3 BCR signaling and cell cycle analysis 85
5.4 Specificity of cell phenotyping 86
6. Conclusion 86
Acknowledgements 86
Referenc es 87
8. First Responders: Laboratory Methods to Assess Human Neutrophils 89
1. Introduction 89
2. One-step Isolation of PMNs by Dextran Sedimentation 91
2.1 Materials 91
2.2 Methods 92
2.3 Notes 92
3. Functional Assays of PMN Function 93
3.1 Detection of surface activation markers and intracellular cytokines by flow cytometry 93
3.1.1 Materials 93
3.1.2 Methods 93
3.1.3 Notes 95
3.2 Analysis of PMN apoptosis by flow cytometry 95
3.2.1 Materials 95
3.2.2 Methods 96
3.2.3 Notes 96
3.3 Detection of specific protein in PMNs by immunoblot 96
3.3.1 Materials 96
3.3.2 Methods 97
3.3.3 Notes 97
3.4 Detection of gene expression in PMN by quantitative PCR (Q-PCR) 98
3.4.1 Materials 98
3.4.2 Methods 98
3.4.3 Notes 99
3.5 Detection of PMN secretion of cytokines or chemokines by ELISA 99
3.5.1 Materials 99
3.5.2 Methods 99
3.5.3 Notes 100
4. Multidimensional Profiling of PMN Immune Status 100
Acknowledgements 100
References 101
9. Multiplexed Transcriptomic Profiling Using Color-Coded Probe Pairs 103
1. Introduction 104
2. The Color-Coded Probe Pair Principle and Applications 104
3. Materials and Methods 107
3.1 12 nCounter gene expression hybridizations 107
3.1.1 Materials 107
3.2 Instruments 109
3.3 Methods 109
3.3.1 Hybridization Step 109
4. 12 nCounter miRNA Expression Hybridizations 110
4.1 Materials 110
4.2 Instruments 110
4.3 Methods 110
4.3.1 Ligation Step 110
4.3.2 Hybridization Step 112
5. Magnetic Bead Purification 112
5.1 Materials 112
5.2 Instruments 112
5.3 Methods 113
6. Data Normalization 113
References 115
10. Statistical Analysis of Human Immunologic Studies: Mixed Effects Modeling 117
1. Introduction 117
2. Repeated Observation Studies 118
2.1 Data visualization 118
2.2. Correlations among repeated measures (covariance structures) 119
2.3. Fixed and random variables and effects 122
2.4. Mixed effects models 123
2.5. Generalized estimating equation models 123
2.6. Checking model fit 124
2.6.1. Fit criteria 124
3. Examples (Mixed Effects Models) 125
3.1 Repeated measures — cross-sectional 125
3.2. Repeated measures — longitudinal (over time) 126
4. Multiple Comparisons 129
4.1. Many separate models versus a unified model 129
5. Summary 131
Acknowledgements 132
References 132
11. Systems Approaches to Autoimmune Diseases 135
1. Introduction 135
2. Decoding Complexity in Autoimmune Disease 137
2.1. Molecular target discovery via network analysis 137
2.2 Extracting cell-specific information from heterogeneous samples 140
3. Challenges and Perspectives 144
References 146
12. Genetic Mapping of Human Immune System Function 151
1. Introduction 151
2. Monogenic Diseases of the Immune System 153
3. Genetic Mapping in Infectious Diseases 156
4. Genetic Mapping of Immune Function and Response Phenotypes 157
5. Conclusions 158
Glossary 159
References 160
Index 165